Binding modes of aromatic ligands to mammalian heme peroxidases with associated functional implications: crystal structures of lactoperoxidase complexes with acetylsalicylic acid, salicylhydroxamic acid, and benzylhydroxamic acid

Abstract

The binding and structural studies of bovine lactoperoxidase with three aromatic ligands, acetylsalicylic acid (ASA), salicylhydoxamic acid (SHA), and benzylhydroxamic acid (BHA) show that all the three compounds bind to lactoperoxidase at the substrate binding site on the distal heme side. The binding of ASA occurs without perturbing the position of conserved heme water molecule W-1, whereas both SHA and BHA displace it by the hydroxyl group of their hydroxamic acid moieties. The acetyl group carbonyl oxygen atom of ASA forms a hydrogen bond with W-1, which in turn makes three other hydrogen-bonds, one each with heme iron, His-109 N(epsilon2), and Gln-105 N(epsilon2). In contrast, in the complexes of SHA and BHA, the OH group of hydroxamic acid moiety in both complexes interacts with heme iron directly with Fe-OH distances of 3.0 and 3.2A respectively. The OH is also hydrogen bonded to His-109 N(epsilon2) and Gln-105N(epsilon2). The plane of benzene ring of ASA is inclined at 70.7 degrees from the plane of heme moiety, whereas the aromatic planes of SHA and BHA are nearly parallel to the heme plane with inclinations of 15.7 and 6.2 degrees , respectively. The mode of ASA binding provides the information about the mechanism of action of aromatic substrates, whereas the binding characteristics of SHA and BHA indicate the mode of inhibitor binding.

FIGURE 1.

A, results showing ASA fitted into 1.5 σ ( F_o − F_c ) electron density. A few residues in the proximity are also indicated. The W-1 corresponds to W-618 in 3GCL. B, SHA fitted into 1.5 σ ( F_o − F_c ) electron density. C, SHA fitted into 1.5 σ ( F_o − F_c ) electron density. The heme moiety, distal Gln-105, and His-109 are also shown. Figures were drawn using Pymol (55).

FIGURE 2.

Activity of lactoperoxidase in the native protein (i), in the presence of ASA (ii), in the presence of SHA (iii), and in the presence of BHA (iv) using ABTS as substrate. The protein ligand molar ratios were 1: 1.

FIGURE 3.

A, the overall folding of the protein shown as a schematic, where α-helices are indicated as cylinders (red) and numbered. H2a is a unique α-helix (yellow) present only in LPO but absent in MPO. The two anti-parallel β-strands are drawn as arrows (blue). For sake of clarity, only ASA (magenta) is shown in the substrate binding site. Heme iron (Fe) is shown in black, whereas conserved heme water molecule (W-1) is indicated in red. The heme moiety is not shown to avoid crowding. Some of the residues, His-109, Phe-113, Phe-380, Gln-423, and Pro-424, of the substrate binding site are shown in ball and stick representation. An arrow indicates the entrance to the substrate binding site. B, close-up view of ligands bound at the distal site ASA (i), SHA (ii), and BHA (iii). The heme iron atom is indicated in green. The ligands are represented as ball and stick models.

FIGURE 4.

Binding of ASA at the distal heme cavity of LPO. The water molecules W-1 and W-3′ correspond to W-618 and W-829, respectively, in Protein Data Bank code 3GCL. The assignment of five subsites has also been shown.

FIGURE 5.

Binding of SHA (yellow) at the distal heme cavity of LPO. The H-bonded interactions are shown as dotted lines. The water molecules W-3′ and W-6′ correspond to W-646 and W-774, respectively, in Protein Data Bank code 3GCJ.

FIGURE 6.

Binding of BHA (yellow) at the distal heme cavity of LPO. The H-bonded interactions are shown as dotted lines. The water molecule W-3′ corresponds to W-772 in Protein Data Bank code 3GCK.